This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background
Picture 1: IRAK 4 and its function in the Cell
This image shows IRAK4 and its role in cell signaling. In image a, IRAK4 helps initiate the cell response to regulate innate immunity. Image b is also an example of IRAK4 catalyzing an immunity signaling pathway (Clark et al 2013). Image from Nature.com.
IRAK 4 (Interleukin 1 receptor (IL-1R)-associated kinase-4) plays a role in the activation of toll-like receptors (TLR) and T-cell receptors (TCR). The IRAK 4 gene also induces the recognition of microbial pathogens through the triggering of innate immune responses. These innate immune responses activate the movement and function of white blood cells in the human body (Aderem & Ulevich et al. 2000).
Even though the kinase activity of the IRAK family members and their molecular mechanism remain unknown (Lye & Mirtsos et al. 2004), we do know that IRAK 4 gene is essential for the signaling pathways leading to mitogen-activated protein kinases. The ability of the IRAK 4 mutant to mediate signals, not all, indicates that it is necessary to take part in the “IL-1R cascade” (Lye & Mirtsos et al. 2004) and is capable of transmitting signal to dependent or independently of its kinase activity.
Methods
The sequence for the assigned protein was obtained from the Ensembl protein database in a FASTA format. Through a BLAST in the whaleshark.georgiaaquarium.org Galaxy server, the human protein sequence (ENSP00000390651) was queried against the whale shark protein database. The five top protein hits were then crosschecked as queries in BLAST searches against the human protein database in NCBI to determine which were the closest matches. The alignment lengths, E-Values, Query Coverages, and Percent Identities were used to determine the closest matches. The IRAK4 predicted orthologs were obtained for Bottle-nose Dolphins, Elephant Sharks, Zebra Fish, and Dogs. The Human protein FASTA was used as the query when performing the BLASTS for the mentioned organisms in NCBI. The best hit for each organism and each whale shark protein BLAST were used to create a phylogenetic tree through ClustalW.
Leucine-rich repeat-containing protein 16B isoform X1
g46616.t1
2e-04
54
693
NACHT, LRR and PYD domains-containing protein 12 isoform X4
This table displays the top 5 potential protein hits that were obtained from the Whale Shark genome when the Human IRAK4 gene was searched.
In order to search for the IRAK4 gene in the Whale Sharks’ genome, a coded amino acid sequence of the IRAK4 protein, or FASTA, was placed into the Georgia aquarium’s Galaxy website. The Galaxy website is a computer program able to search for similar amino acid sequences in the Whale Shark’s genome. The Galaxy site returned five possible Whale Shark sequences that matched part of the Human IRAK4 Protein. These 5 sequences and their E-values are listed in the Table 1 above. These 5 sequences were cross-checked in the NCBI Blast web site. A potential Whale Shark protein FASTA was placed into NCBI BLAST, and it was searched for in the human sequence. Surprisingly, all five potential proteins did not match back to the protein that IRK4 codes for; they matched different human proteins, leading to the conclusion that IRAK 4 does not have a comparable protein in Whale Sharks.
Orthologues
Picture 2. Picture of the Elephant Shark BLAST Output This is a picture of the search results obtained by performing a BLAST search of the human IRAK4 sequence in the elephant shark database of the NCBI BLAST site. The best protein match is shown by the red line and greater than 200 amino acids match between the two sequences. The protein also belongs to the Death Domain Superfamily.
Since, the Human FASTA sequence for IRAK4 did not return any matches with the whale shark sequence, the sequence was compared to Elephant Sharks next because they are closely related to Whale Sharks. In fact, the IRAK 4 gene did have an orthologue in the Elephant Shark sequence. A BLAST with the IRAK FASTA sequence returned a close protein match with an E-value of 8e-70 and a query coverage of 94%, which had the same name (interleukin 1 receptor associated kinase), and also was part of the Death Domain Superfamily. These pieces of evidence led to the conclusion that the IRAK4 gene has an orthologue with the Elephant Shark.
Protein Domains Picture 1: Picture of IRAK4 Protein Domain in Humans This is an image of the IRAK4 domains shown by the NCBI BLAST site.
The IRAK4 gene belongs to the Death Domain Superfamily and the Catalytic domain of the Serine/Threonine kinase (STK). A protein domain is a subunit of the protein that is present in tertiary structure and able to perform a task that contributes to the overall function of the protein. Two different proteins can have a similar domain in part of their sequences ("What are Protein Domains?" 2015). Death Domain proteins play a role in signaling pathways, especially cell death (Marchler-Bauer 2015). STKs help transfer the gamma-phosphoryl group from ATP to serine/threoinine substrates on a protein (Marchler-Bauer 2015).
Phylogeny Picture 3. Phylogenetic tree of IRAK4 and Related Organisms This phylogenetic tree displays the relationship between the Whale Shark proteins, the Zebrafish, the Elephant Shark, Humans, Dogs, and the Bottle-nosed Dolphin. Notice that there is a divergence from the Whale Shark proteins and the other species. The other species do not show much resemblance with the Whale Shark, which suggests that this protein is not an exact match in the Whale Shark, but has changed. The human protein is more closely related to other species such as dogs, bottle-nosed dolphins and zebrafish. As the tree indicates, the Elephant Shark Protein is closer related to the Human Protein than any of the Whale Shark Potential Proteins.
Conclusions
It seems that at some point in time the IRAK4 gene was lost between the Elephant Shark and the Whale Shark species. This conclusion was reached because there were no definite protein matched between the Human protein and the Whale Shark Protein. The lack of an exact protein is evident in Table 1. However, there was as orthologues found in between the Human and Elephant Shark. This is particularly interesting because the Whale Shark and the Elephant Shark are closely related. The IRAK4 protein was conserved between Elephant Sharks and Humans, but not between Elephant Sharks and Whale Sharks. It would be interesting to further investigate how the loss of the IRAK4 gene affects the performance and or development of the Whale Shark. It could be supposed that Whale Sharks have a different mechanism to trigger white blood cells as an innate immune system response. The IRAK4 gene will continue to be studied for more thorough conclusions as the "whys" and "hows" we have drawn up from our current research.
References
Aderem, Alan, and Richard J. Ulevitch. "Toll-like Receptors in the Induction of the Innate Immune Response." Nature: International Weekly Journal of Science. N.p., 17 Aug. 2000. Web. 30 Mar. 2015.
Lye, Elizabeth, Christine Mirtsos, Nobutaka Suzuki, Shinobu Suzuki, and Wen-Chen Yeh. "The Role of Interleukin 1 Receptor-associated Kinase-4 (IRAK-4) Kinase Activity in IRAK-4-mediated Signaling." The Role of Interleukin 1 Receptor-associated Kinase-4 (IRAK-4) Kinase Activity in IRAK-4-mediated Signaling. The Journal of Biological Chemistry, 9 Mar. 2004. Web. 30 Mar. 2015.
Clark K, Nanda S, Cohen P. "Molecular Controls of the NEMO family ubiquitin-binding proteins". Nature Reviews: Molecular Cell Biology. 14, 673-685. 29 August 2013. Wed. 6 April 2015. <http://www.nature.com/nrm/journal/v14/n10/full/nrm3644.html>.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43 (Database issue): D222-6
Uematsu, Satoshi, Shintaro Sato, Masahiro Yamamoto, Tomonori Hirotani, Hiroki Hato, Fumihiko Takeshita, Michiyuki Matsuda, Cevayir Coban, Ken J. Ishii, Taro Kawai, Osamu Takeuchi, and Shizuo Akira. "Interleukin-1 Receptor-associated Kinase-1 Plays an Essential Role for Toll-like Receptor (TLR)7- and TLR9-mediated Interferon-α Induction." Interleukin-1 Receptor-associated Kinase-1 Plays an Essential Role for Toll-like Receptor (TLR)7- and TLR9-mediated Interferon-α Induction. The Journal of Experimental Medicine, 14 Mar. 2005. Web. 30 Mar. 2015.
This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.Background
Picture 1: IRAK 4 and its function in the Cell
This image shows IRAK4 and its role in cell signaling. In image a, IRAK4 helps initiate the cell response to regulate innate immunity. Image b is also an example of IRAK4 catalyzing an immunity signaling pathway (Clark et al 2013). Image from Nature.com.
IRAK 4 (Interleukin 1 receptor (IL-1R)-associated kinase-4) plays a role in the activation of toll-like receptors (TLR) and T-cell receptors (TCR). The IRAK 4 gene also induces the recognition of microbial pathogens through the triggering of innate immune responses. These innate immune responses activate the movement and function of white blood cells in the human body (Aderem & Ulevich et al. 2000).
Even though the kinase activity of the IRAK family members and their molecular mechanism remain unknown (Lye & Mirtsos et al. 2004), we do know that IRAK 4 gene is essential for the signaling pathways leading to mitogen-activated protein kinases. The ability of the IRAK 4 mutant to mediate signals, not all, indicates that it is necessary to take part in the “IL-1R cascade” (Lye & Mirtsos et al. 2004) and is capable of transmitting signal to dependent or independently of its kinase activity.
Methods
The sequence for the assigned protein was obtained from the Ensembl protein database in a FASTA format. Through a BLAST in the whaleshark.georgiaaquarium.org Galaxy server, the human protein sequence (ENSP00000390651) was queried against the whale shark protein database. The five top protein hits were then crosschecked as queries in BLAST searches against the human protein database in NCBI to determine which were the closest matches. The alignment lengths, E-Values, Query Coverages, and Percent Identities were used to determine the closest matches. The IRAK4 predicted orthologs were obtained for Bottle-nose Dolphins, Elephant Sharks, Zebra Fish, and Dogs. The Human protein FASTA was used as the query when performing the BLASTS for the mentioned organisms in NCBI. The best hit for each organism and each whale shark protein BLAST were used to create a phylogenetic tree through ClustalW.
Searching the Whale Shark Genome for IRAK4
Table 1. Possible Whale Shark IRAK4 Matches
In order to search for the IRAK4 gene in the Whale Sharks’ genome, a coded amino acid sequence of the IRAK4 protein, or FASTA, was placed into the Georgia aquarium’s Galaxy website. The Galaxy website is a computer program able to search for similar amino acid sequences in the Whale Shark’s genome. The Galaxy site returned five possible Whale Shark sequences that matched part of the Human IRAK4 Protein. These 5 sequences and their E-values are listed in the Table 1 above. These 5 sequences were cross-checked in the NCBI Blast web site. A potential Whale Shark protein FASTA was placed into NCBI BLAST, and it was searched for in the human sequence. Surprisingly, all five potential proteins did not match back to the protein that IRK4 codes for; they matched different human proteins, leading to the conclusion that IRAK 4 does not have a comparable protein in Whale Sharks.
Orthologues
Picture 2. Picture of the Elephant Shark BLAST Output
This is a picture of the search results obtained by performing a BLAST search of the human IRAK4 sequence in the elephant shark database of the NCBI BLAST site. The best protein match is shown by the red line and greater than 200 amino acids match between the two sequences. The protein also belongs to the Death Domain Superfamily.
Since, the Human FASTA sequence for IRAK4 did not return any matches with the whale shark sequence, the sequence was compared to Elephant Sharks next because they are closely related to Whale Sharks. In fact, the IRAK 4 gene did have an orthologue in the Elephant Shark sequence. A BLAST with the IRAK FASTA sequence returned a close protein match with an E-value of 8e-70 and a query coverage of 94%, which had the same name (interleukin 1 receptor associated kinase), and also was part of the Death Domain Superfamily. These pieces of evidence led to the conclusion that the IRAK4 gene has an orthologue with the Elephant Shark.
Protein Domains
Picture 1: Picture of IRAK4 Protein Domain in Humans
This is an image of the IRAK4 domains shown by the NCBI BLAST site.
The IRAK4 gene belongs to the Death Domain Superfamily and the Catalytic domain of the Serine/Threonine kinase (STK). A protein domain is a subunit of the protein that is present in tertiary structure and able to perform a task that contributes to the overall function of the protein. Two different proteins can have a similar domain in part of their sequences ("What are Protein Domains?" 2015). Death Domain proteins play a role in signaling pathways, especially cell death (Marchler-Bauer 2015). STKs help transfer the gamma-phosphoryl group from ATP to serine/threoinine substrates on a protein (Marchler-Bauer 2015).
Phylogeny
Picture 3. Phylogenetic tree of IRAK4 and Related Organisms
This phylogenetic tree displays the relationship between the Whale Shark proteins, the Zebrafish, the Elephant Shark, Humans, Dogs, and the Bottle-nosed Dolphin. Notice that there is a divergence from the Whale Shark proteins and the other species. The other species do not show much resemblance with the Whale Shark, which suggests that this protein is not an exact match in the Whale Shark, but has changed. The human protein is more closely related to other species such as dogs, bottle-nosed dolphins and zebrafish. As the tree indicates, the Elephant Shark Protein is closer related to the Human Protein than any of the Whale Shark Potential Proteins.
Conclusions
It seems that at some point in time the IRAK4 gene was lost between the Elephant Shark and the Whale Shark species. This conclusion was reached because there were no definite protein matched between the Human protein and the Whale Shark Protein. The lack of an exact protein is evident in Table 1. However, there was as orthologues found in between the Human and Elephant Shark. This is particularly interesting because the Whale Shark and the Elephant Shark are closely related. The IRAK4 protein was conserved between Elephant Sharks and Humans, but not between Elephant Sharks and Whale Sharks. It would be interesting to further investigate how the loss of the IRAK4 gene affects the performance and or development of the Whale Shark. It could be supposed that Whale Sharks have a different mechanism to trigger white blood cells as an innate immune system response. The IRAK4 gene will continue to be studied for more thorough conclusions as the "whys" and "hows" we have drawn up from our current research.
References
Aderem, Alan, and Richard J. Ulevitch. "Toll-like Receptors in the Induction of the Innate Immune Response." Nature: International Weekly Journal of Science. N.p.,
17 Aug. 2000. Web. 30 Mar. 2015.
Lye, Elizabeth, Christine Mirtsos, Nobutaka Suzuki, Shinobu Suzuki, and Wen-Chen Yeh. "The Role of Interleukin 1 Receptor-associated Kinase-4 (IRAK-4) Kinase Activity in IRAK-4-mediated Signaling." The Role of Interleukin 1 Receptor-associated Kinase-4 (IRAK-4) Kinase Activity in IRAK-4-mediated Signaling. The Journal of Biological Chemistry, 9 Mar. 2004. Web. 30 Mar. 2015.
Clark K, Nanda S, Cohen P. "Molecular Controls of the NEMO family ubiquitin-binding proteins". Nature Reviews: Molecular Cell Biology. 14, 673-685. 29 August 2013. Wed. 6 April 2015. <http://www.nature.com/nrm/journal/v14/n10/full/nrm3644.html>.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43 (Database issue): D222-6
Uematsu, Satoshi, Shintaro Sato, Masahiro Yamamoto, Tomonori Hirotani, Hiroki Hato, Fumihiko Takeshita, Michiyuki Matsuda, Cevayir Coban, Ken J. Ishii, Taro Kawai, Osamu Takeuchi, and Shizuo Akira. "Interleukin-1 Receptor-associated Kinase-1 Plays an Essential Role for Toll-like Receptor (TLR)7- and TLR9-mediated Interferon-α Induction." Interleukin-1 Receptor-associated Kinase-1 Plays an Essential Role for Toll-like Receptor (TLR)7- and TLR9-mediated Interferon-α Induction. The Journal of Experimental Medicine, 14 Mar. 2005. Web. 30 Mar. 2015.
"What Are Protein Domains?" The European Bioinformatics Institute. European Molecular Biology Laboratory, 2015. Web. 6 April 2015. <
https://www.ebi.ac.uk/training/online/course/introduction-protein-classification-ebi/protein-classification/what-are-protein-domains>.